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TitleLigand Incorporation into Protein Microcrystals for MicroED by On-Grid Soaking.
Journal, issue, pagesStructure, Vol. 29, Issue 1, Page 88-95.e2, Year 2021
Publish dateJan 7, 2021
AuthorsMichael W Martynowycz / Tamir Gonen /
PubMed AbstractA high throughout method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoelectron microscopy ...A high throughout method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoelectron microscopy vitrification equipment. The method is demonstrated using proteinase K microcrystals soaked with the 5-amino-2,4,6-triodoisophthalic acid (I3C) magic triangle. A soaked microcrystal is milled to a thickness of approximately 200 nm using a focused ion beam, and MicroED data are collected. A high-resolution structure of the protein with four ligands at high occupancy is determined. Both the number of ligands bound and their occupancy is higher using on-grid soaking of microcrystals compared with much larger crystals treated similarly and investigated by X-ray crystallography. These results indicate that on-grid soaking ligands into microcrystals results in efficient uptake of ligands into protein microcrystals.
External linksStructure / PubMed:33007196 / PubMed Central
MethodsEM (electron crystallography)
Resolution1.78 Å
Structure data

22463

7jsy

EMDB-22463, PDB-7jsy:
Proteinase K soaked with I3C determined by MicroED from a single milled microcrystal
Method: EM (electron crystallography) / Resolution: 1.78 Å

Chemicals

ChemComp-I3C:
5-amino-2,4,6-triiodobenzene-1,3-dicarboxylic acid

ChemComp-HOH:
WATER / Water

Source
  • parengyodontium album (fungus)
KeywordsHYDROLASE / Proteinase K

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